Thermolysin [EC 3.4.24.27; CAS registry number 9073-78-3] is a thermostable neutral metalloproteinase (also referred to herein as “neutral protease”) produced in the culture broth of Bacillus thermoproteolyticus (Endo, S., J., Ferment. Technol. 40 (1962) 346-353; Matsubara, H., Feder, J., in: 3rd ed., Boyer, P., D., (Ed.), The Enzymes, vol. 3, Academic Press, New York, 1971, pp. 721-795). It requires one zinc ion for enzyme activity and four calcium ions for structural stability (Latt, S., A., et al. Biochem. Biophys. Res. Commun. 37 (1969) 333-339; Feder, J., et al. Biochemistry 10 (1971) 4552-4556; Tajima, M., et al. Eur. J. Biochem. 64 (1976) 243-247) and catalyzes specifically the hydrolysis of peptide bonds containing hydrophobic amino acid residues (Morihara, K., Tsuzuki, H., Eur. J. Biochem. 15 (1970) 374-380; Inouye, K., et al. Biochem. J. 315 (1996) 133-138). Thermolysin is widely used for the peptide bond formation through reverse reaction of hydrolysis (Oyama, K., et al., J. Chem. Soc. Perkin II (1981) 356-360; Nakanishi, K., et al., Ann. N.Y. Acad. Sci. 613 (1990) 652-655; Trusek-Holownia, A., J. Biotechnol. 102 (2003) 153-163). The npr gene that encodes thermolysin was isolated from B. thermoproteolyticus (O'Donohue, M., J., et al., Biochem. J. 300 (1994) 599-603). Sequence analysis reveals that thermolysin is synthesized as a pre-proprotein consisting a signal peptide (28 residues), a prosequence (204 residues), and a mature sequence (316 residues) (O'Donohue, M., J., et al., supra). The prosequence acts as an intramolecular chaperone leading to an autocatalytic cleavage of the peptide bond linking the pro and mature sequences (O'Donohue, M., J., et al., J. Biol. Chem. 271 (1996) 26477-26481; Marie-Claire, C., et al., J. Biol. Chem. 273 (1998) 5697-5701; Marie-Claire, C., et al., J. Mol. Biol. 285 (1999) 1911-1915).
The theoretical extinction at 280 nm of intact thermolysin in water can be calculated using the “ProtParam tool” which is publicly available via the internet. ProtParam is a tool which allows the computation of various physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or for a user entered sequence. The computed parameters include the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity. Accordingly, the theoretical absorbance value in water of A (1 mg/ml), at 280 nm of 1.696 can be calculated.
The manufacturer of thermolysin (Daiwa Kasei K.K., Japan) referring to Ohta, Y et al. (J. Biol. Chem. 241 (1966) 5919-5925) indicates an absorbance of A (1 mg/ml), of 1.765 at 280 nm in 50 mM TrisHCl buffer, pH 7.
Inouye, K., et al. (J. Biochem. 123 (1998) 847-852) discloses an absorbance value A (1 mg/ml) of 1.83, determined at 277 nm and 25° C. for thermolysin Lot T8BA51 (Daiwa Kasei K.K., Osaka, Japan) in 10 mM CaCl2, 40 mM TrisHCl, pH 7.5.
Thermolysin can be obtained as a lyophilisate from commercial suppliers. Daiwa Kasei K.K. (Japan) distributes a thermolysin with a molecular weight of 34,600 Da (Daltons), a pH optimum at pH 8.0, and a temperature optimum in the range of 65° C. and 70° C. According to the manufacturer, the enzyme is stable in a pH range of pH 5.0 and pH 8.5. A solubility of 0.02% in dilute buffer solution is indicated. Twice crystallized thermolysin can be purchased as a freeze-dried amorphous powder, wherein the enzyme protein in the dried matter is 60% [w/w] or higher. The dried matter additionally contains anhydrous calcium acetate (about 20% [w/w]) and anhydrous sodium acetate (about 10% [w/w]). For further crystallization the manufacturer describes a method comprising the steps of suspending the lyophilisate at a concentration in the range of 1% [w/v] and 5% [w/v] in an aqueous solution of 0.01 M Calcium-acetate. The suspended material is dissolved by adding drop wise enough 0.2 N sodium hydroxide under agitation to bring the pH of the aqueous solution to a value in the range of pH 11.0 and pH 11.4. After removal of any undissolved residue, the pH of the solution is adjusted to pH 6.0 with 0.2 N acetic acid. The crystallization is usually complete in approximately 2 days. The whole process is performed at a temperature in the range of about 0° and 2° C.
Preparations of thermolysin are also available from Daiwa Kasei K.K. (Japan) under the trade name Thermoase.
EP 0 640 687 discloses an aqueous solution of 7 mM CaCl2 and 1.75 M NaCl in which Thermoase was dissolved to result in a concentration of about 36 mg/ml. The purity of thermolysin in the dry Thermoase powder was about 20%. Taking purity into account, the concentration of thermolysin in the aqueous solution was about 7 mg/ml.
Inouye, K., et al. (J. Biochem. 123 (1998) 847-852) reported that thermolysin is a sparingly soluble protein. It was suggested that the surface of the protein is hydrophobic to a large extent and the fact that thermolysin can be purified efficiently by way of hydrophobic interaction chromatography supports this notion (Inouye, K., et al., Protein Expression and Purification 46 (2006) 248-255). As a consequence of its low solubility, the enzyme has a strong tendency to precipitate within hours from freshly prepared solutions.
Inouye, K. et al. (J. Biochem. supra) further demonstrated that the solubility of thermolysin in an aqueous solvent can be increased if certain neutral salts are dissolved in the solvent when it is contacted with a lyophilized preparation of thermolysin. The effect was shown to be dependent on (1) temperature, (2) the particular neutral salt present in the solvent, and (3) the concentration of the respective neutral salt. In the document of Inouye, K. et al. (J. Biochem. supra) FIG. 2 discloses the results of a series of experiments in which an excessive amount of thermolysin lyophilized powder (three-times-crystallized-and-lyophilized preparation of thermolysin (Daiwa Kasei K.K., Osaka, Japan; Lot T8BA51; used without further purification) was mixed with a “standard buffer” (10 mM CaCl2, 40 mM TrisHCl, pH 7.5) which additionally contained a salt at a predetermined concentration (in the range of 0.5 M and 5 M). The concentration of dissolved protein was determined spectrophotometrically using an absorbance value, A (1 mg/ml), at 277 nm of 1.83 and a molecular mass of 34.6 kDa.
Tables 1-4 reproduce the approximate numerical values indicating the concentrations of dissolved protein as graphically depicted in FIG. 2 of Inouye, K. et al. (J. Biochem. supra), at two different temperatures (0° C. and 37° C.). The protein concentrations tabulated are in mg/ml. In each table the salts dissolved in the standard buffer are indicated as well as their respective concentrations.
TABLE 1Concentrations of protein (in [mg/ml]) soluble at 0° C. in standardbuffer containing saltconcentration of salt instandard buffer0.5M1.0M1.5M2.0M2.5MsaltNaCl~6.4~8.9~10.3~12.2~11.6KCl~4.5~6.3~7.5~6.5~5.3LiCl~1.9~3.3~4.5~5.3~6.6NaBr~4.4~6.6~15.6~25.3~38.4NaJ~5.5~7.8~20.3~29.2~32.5
TABLE 2Concentrations of protein (in [mg/ml]) soluble at 0° C. in standardbuffer containing saltconcentration of saltin standard buffer3.0M3.5M4.0M4.5M5.0MsaltNaCl~9.5~8.1~6.9~5.3~3.8KCl~5.2~4.4~4.1~3.4~2.5LiCl~8.0~11.1~14.7~21.6~26.3NaBr~40~36.6~30~27.8~25.3NaJ~34.4~37.5~38.6~42.3§§ out of detection range
TABLE 3Concentrations of protein (in [mg/ml]) soluble at 37° C. in standardbuffer containing saltconcentration of saltin standard buffer0.5M1.0M1.5M2.0M2.5MsaltNaCl~2.7~3.8~5.5~7.5~8.8KCl~1.6~3.1~3.2~4.2~3.4LiCl~0.9~2.2~2.5~3.1~4.5NaBr~3.4~5.0~9.7~13.3~18NaJ~3.4~6.2~18~24.5~34.4
TABLE 4Concentrations of protein (in [mg/ml]) soluble at 37° C. in standardbuffer containing saltconcentration of saltin standard buffer3.0M3.5M4.0M4.5M5.0MsaltNaCl~7.4~5.5~3.1~2.3~1.0KCl~3.1~2.8~2.2~1.9~0.6LiCl~7.0~8.8~11.3~18~22.3NaBr~22.2~24.4~26.3~29~33.1NaJ~36.7~38.1~35.3~38.6~38.0
Accordingly, for selected salts the highest concentrations of soluble protein were each about                8.8 mg/ml at 37° C. in the presence of 2.5 M NaCl,        12.2 mg/ml at 0° C. in the presence of 2 M NaCl,        4.2 mg/ml at 37° C. in the presence of 2 M KCl,        7.5 mg/ml at 0° C. in the presence of 1.5 M KCl,        22.3 mg/ml at 37° C. in the presence of 5 M LiCl,        26.3 mg/ml at 0° C. in the presence of 5 M LiCl,        33.1 mg/ml at 37° C. in the presence of 5 M NaBr,        40 mg/ml at 0° C. in the presence of 3 M NaBr,        38.6 mg/ml at 37° C. in the presence of 4.5 M NaJ, and        >45 mg/ml at 0° C. in the presence of 5 M NaJ.        
Thermolysin is an aggressive protease which in solution undergoes autoproteolytic attack. Thus, both crystallized and freeze-dried preparations of thermolysin as well as solutions of such preparations contain amounts of different autoproteolytic fragments of thermolysin.
In order to limit autoproteolytic attack, low temperatures are applied to solutions containing thermolysin. However, enzymatic activity is only reduced (i.e. some proteolytic activity is still present) under such conditions, and not brought to a complete halt. In this regard it is noted that Inouye, K. et al. (J. Biochem. supra) determines protein content of solutions without any purification step. The protein concentrations detected therefore correspond to mixtures of intact thermolysin and degradation fragments thereof.
In view of the state of the art it is an object of the present invention to provide methods and compositions with a stabilized form of thermolysin in an aqueous solution. By providing a stabilized form, the tendency of thermolysin to precipitate is reduced, and solutions of the enzyme remain in a homogeneous state for a prolonged time.
The inventors have unexpectedly found that by dissolving thermolysin first in a buffer with a low ion concentration and then adding a salt and dissolving the salt in the solution which already contains thermolysin surprisingly allows to form a solution with a high concentration of thermolysin. At the same time, under such conditions according to the invention dissociated thermolysin is stabilized in the solution, i.e. the solution remains clear for an increased amount of time during which no precipitate is formed.
The invention provides significant benefit when amounts of thermolysin have to be kept in solution for dispensing aliquots thereof, or for making blends with preparations of other enzymes, such as collagenase enzymes. Such blends of proteolytic enzymes are of particular use in the dissociation of organ tissue for the separation of subsets of cells from the tissue.